Administration of interleukin-12 exerts a therapeutic instead of a long-term preventive effect on mite Der p I allergen-induced animal model of airway inflammation

Abstract

Interleukin-12 (IL-12) is a key cytokine, which promotes T helper type 1 (Th1) cell-mediated immunity and inhibits Th2-type responses. It has been previously shown that IL-12 administration during active immunization following a single allergen exposure can prevent antigen-induced increases in immunoglobulin E (IgE) formation, Th2 cytokine production and bronchoalveolar lavage (BAL) eosinophils in a murine model of allergic airway inflammation. Thus, these studies have now been extended and two IL-12 treatment protocols on this murine model were evaluated. Administration of IL-12 during the active immunization strikingly increased Der p I-specific serum IgG2a and transiently decreased the levels of IgG1 and IgE antibodies following multiple allergen challenges. Such early treatment of IL-12 down-regulated IL-5 production and modestly up-regulated interferon-gamma production but did not effect BAL eosinophilia. These results suggest that repeated exposure to antigen and IL-12 is necessary to maintain a persistent Th1-recall response. Furthermore, administration of IL-12 to actively immunized mice, in which Th2-associated responses were established, had a significant effect on IgG2a synthesis and a modest effect on IgE levels, also down-regulation of IL-5 production, and markedly increased interferon-gamma production and abolished recruitment of eosinophils. Therefore, these data indicate that IL-12 can inhibit antigen-induced eosinophil infiltration into airways, despite the existence of a Th2-associated response. Taken together, these studies suggest that IL-12 may be useful as an immunotherapeutic agent in the treatment of such pulmonary allergic disorders as bronchial asthma.

Figure 1

Immunization protocol of the four experimental groups of C57BL/6 mice (n =8). Intraperitoneal (i.p.) Der p I injections consisted of 10 μg of Der p I, 2 mg Al(OH)₃ and 400 ng pertussis toxin dissolved in 100 μl of PBS/dose. Dp inhalation was performed with 8 mg of Dp dissolved in 8 ml of PBS/time. Intraperitoneal IL-12 consisted of 1 μg of IL-12 dissolved in 50 μl of PBS/dose and treated mice for 5 days (day −1 to +3) simultaneously with immunization indicated; i.p. PBS consisted of 50 μl of PBS/dose.

Figure 2

Serum IgG1, IgE and IgG2a antibody responses to Der p I. Groups 1 and 2 are described in the Materials and Methods. C57BL/6 mice were immunized i.p. three times with 10 μg/mouse Der p I adsorbed to alum plus pertussis toxin at biweekly intervals. In Group 2, IL-12 was administered i.p. at 1 μg/day for 5 days (day −1 to +3) to mice simultaneously with each systemic immunization. After 2 weeks, sensitized mice were aerosolized three times with Dp extract on days 42, 56 and 70. Blood was collected on the days indicated and sera levels of anti-Der p I antibodies IgG1 (a), IgE (b) and IgG2a (c) were assayed by using ELISA. Data are shown as mean±SEM for eight mice per group. Significant differences (*P < 0·05; ***P < 0·001) from the immunized IL-12-untreated Group 1 are indicated.

Figure 3

Serum IgG1, IgE and IgG2a antibody responses to Der p I. Groups 3 and 4 are described in the Materials and Methods. C57BL/6 mice were immunized i.p. four times with 10 μg/mouse Der p I adsorbed to alum plus pertussis toxin at biweekly intervals. In Group 4, IL-12 was administered i.p. at 1 μg/day for 5 days (day −1 to +3) to mice simultaneously with the third and fourth systemic immunizations. After 2 weeks, each group of sensitized mice was challenged with aerosolized Dp allergen. Blood was collected on the days indicated and sera levels of anti-Der p I antibodies IgG1 (a), IgE (b) and IgG2a (c) were assayed by using ELISA. Data are shown as mean±SEM for eight mice per group. Significant differences (*P < 0·05; ***P < 0·001) from the immunized IL-12-untreated Group 3 are indicated.

Figure 4

The effect of IL-12 on Der p I antigen-specific T-cell proliferative response. C57BL/6 mice were treated as described in Fig. 1. Following the last inhalation, spleen cells were taken from these sensitized mice after 24 hr and restimulated with 10 μg/ml Der p I in vitro. Proliferation was measured by [³H]thymidine incorporation on day 3. The results are expressed as c.p.m. and shown as mean±SEM for seven or eight mice per group. The background values of controls were between 1450 and 2534 c.p.m. Ovalbumin (4 μg/ml) was used as a negative control antigen and the responses were between 1045 and 3138 c.p.m. in this assay. The stimulation index (SI) was calculated as the mean c.p.m. of the stimulated wells divided by the mean c.p.m. of the control wells. Significant differences (*P ≤ 0·05; **P ≤ 0·001) from the immunized untreated controls are indicated.

Figure 5

The effect of IL-12 on IFN-γ, IL-5 production by splenocytes of mice after the last allergen challenge. Splenocytes from IL-12 and control groups were stimulated with 10 μg/ml Der p I in vitro and culture supernatants were collected after 48 hr, and the levels of cytokine production IFN-γ (a) and IL-5 (b) were measured by ELISA. Values shown are mean±SD of eight mice per group. Significant differences (*P < 0·05, ***P < 0·001) compared with paired control mice, respectively.

Figure 6

Representative light microscopic findings of PBS-treated mice (a, c) and IL-12-treated mice (b, d) (hematoxylin and eosin stain). Lung tissue of mice without IL-12 treatment (a, ×400, and c, ×200) demonstrates extensive cellular infiltration of the periairway regions. In contrast, lung tissue of IL-12-treated mice (b, ×400, and d, ×200) demonstrates complete absence of histologically significant inflammation.

Figure 7

The effect of IL-12 on serotonin production in BAL of mice after the last antigen challenge. C57BL/6 mice were treated as described in Fig. 1. Mice were challenged with inhaled Dermatophagoides pteronyssinus and BAL samples were taken from the mice with allergen challenged after 24 hr. BAL fluids from each group of mice were measured by using a specific ELISA. Data represent the mean±SD of eight samples per group. **P < 0·05 compared with Group 1; ***P < 0·001 compared with Group 3.